Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.Rapid advances in understanding the molecular changes in human oncogenesis have been provided by transfection of tumor DNAs into NIH 3T3 cells (1). Of all human tumor DNA tested, however, some 70% are ineffective in 3T3 transformation (2). In developing a DNA transfection system using normal human cells as recipients (3), we found that the cells could be transformed to anchorage-independent growth by MOLT-4 DNA, which transforms 3T3 cells inefficiently (4) or not at all (5). We have used a series of three multiple myeloma cell lines, GM 1312, GM 1500, and GM 2132, to examine transformation of human cells by tumor DNAs that do (GM 1500 and GM 2132) and do not (GM 1312) transform NIH 3T3 cells (5).We show that DNA of GM 1312 does transform human cells to anchorage-independent growth, yielding transfectants that grow in soft agar at much higher frequencies than do the parental fibroblasts and whose DNA can transform naive recipients to anchorage-independent growth. DNAs of GM 2132 and GM 1500 transform human cells at a higher frequency than does that from GM 1312. Since GM 1500 DNAs transform both human and 3T3 cells, we tested by restriction analysis whether the same gene was acting in the two systems: although restriction by five enzymes gave similar results in the two systems, Sac I inactivated the transforming activity of the DNA in 3T3 cells (5) but not in human cells, suggesting the possibility that different genes are recognized by the two cellular recipients.These results show that the human cell transfection system can detect transforming genes in DNAs ineffective in the 3T3 system and indicate the possibility of detecting additional transforming sequences in DNAs containing oncogenes that can transform 3T3 cells.
MATERIALS AND METHODSCells. Neonatal human fibroblasts were obtained from primary cultures established in this laboratory with foreskins from Central Suffolk Hospital (Riverhead, NY) or Brookhaven Hospital (Patchogue, NY). Stock cultures were grown without antibiotics in a Dulbecco's modified Eagle's m...
Chronic exposure of the gray, short-tailed oppossum, Monodelphis domestica to ultraviolet radiation (UVR) induces mesenchymal tumors of the cornea. High molecular weight DNA samples from 6 UVR-induced corneal tumors were assayed for their ability to transform NIH 3T3 cells to tumorigenicity. NIH 3T3 cells transfected with DNA from 5 of the corneal tumors produced 14 tumors in nude mice. Cell lines were established from these tumors. DNA from 13 of 14 tumor cell lines contained repetitive opossum DNA sequences. Southern blot analysis revealed that DNA from 3 of 4 cell lines derived from tumorigenic NIH 3T3 cells transfected with DNA from a single oppossum tumor contained opossum Ki-ras oncogene sequences in addition to the murine Ki-ras gene. Northern blot analysis of mRNA from a mouse tumor cell line containing opossum Ki-ras gene sequences showed mRNA species identical in size to opossum Ki-ras mRNA, as well as murine Ki-ras mRNA species. These results suggest that an activated Ki-ras oncogene was present in one of the original opossum corneal tumors tested. Thus, activation of Ki-ras may play a role in the development of UVR-induced corneal tumors in Monodelphis domestica. Further characterization of ras oncogenes in these opossum tumors may provide information on the molecular mechanisms by which UVR induces corneal tumors in this species.
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